Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

Abstract

Hereditary folate malabsorption (HFM) is an autosomal recessive disorder characterized by impaired intestinal folate absorption and impaired folate transport across the choroid plexus due to loss of function of the proton-coupled folate transporter (PCFT-SLC46A1). We report a novel mutation, causing HFM, affecting a residue located in the 11th transmembrane helix within the external gate. The mutant N411K-PCFT was stable, trafficked to the cell membrane, and had sufficient residual activity to characterize the transport defect and the structural requirements at this site for gate function. The influx V_max of the N411K mutant was markedly decreased, as was the affinity for most, but not all, folate/antifolate substrates. The greatest loss of activity was for 5-methyltetrahydrofolate. Substitutions with positive charged residues resulted in a loss of activity (arginine > lysine > histidine). Function was retained for the negative charged aspartate, but not the larger glutamate substitutions, whereas the bulky hydrophobic (leucine), or polar (glutamine) substitutions, were tolerated. Homology models of PCFT, in the inward and outward open conformations, based upon the mammalian Glut5 fructose transporter structures, localize Asn411 protruding into the aqueous pathway. This is most prominent when the carrier is in the inward open conformation when the external gate is closed. Mutations at this site likely result in highly specific steric and electrostatic interactions between the Asn411-substituted, and other, residues in the gate region that impede carrier function. The substrate specificity of the N411K mutant may be due to alterations of substrate flows through the external gate, downstream allosteric alterations in the folate-binding pocket, or both.

Graphical abstract

Figure 1.

Expression and function of the N411K PCFT mutant. (A) Influx of 0.5 μM [³H] MTX was assessed over 1 min at 37°C in RFC-/PCFT-null HeLa R1-11 cells transfected with wild-type PCFT, the N411K mutant, or vector control. Data are indicated as percentage of PCFT-WT activity and are the average of 3 independent experiments ± SEM. (B) Expression at the cell surface is depicted in the upper row. PCFT expression in the crude membrane preparation is depicted in the lower row. The western blot is representative of 3 independent experiments.

Figure 2.

Structural requirement at the Asn411 residue. (A) Expression at the cell surface (upper row), and in the crude membrane fraction (middle row), of the Asn411 mutants. β-Actin was the internal control (lower row). The blot is representative of 3 independent experiments. (B) Influx of 0.5 and 50 μM [³H] MTX was measured in HeLa R1-11 cells transfected with a variety of substitutions at the Asn411 residue. Data are the averages ± SEM of 3 independent experiments. *P < .01; **P < .0001, in comparison with PCFT-WT activity.

Figure 3.

Effects of sulfhydryl modifications of the N411C PCFT mutant. Cells harboring the N411C PCFT mutation were treated with 3 mM MTSES, 3 mM MTSET, or 0.5 mM MTSEA-biotin for 30 min at pH 7.4 and RT followed by assessment of [³H]MTX influx. Data are percentages of untreated cells ± SEM, from the average of 3 independent experiments.

Figure 4.

pH-dependence of [³H]MTX influx. Activities for the PCFT-WT and N411K PCFT mutant are normalized to the rate at pH 5.5, indicated as 100%. Data are the means ± SEM from 3 independent experiments.

Figure 5.

Substrate specificity of transport mediated by the N411K PCFT mutant. Influx of tritiated PCFT substrates was assessed at a concentration far below the influx K_t for PCFT-WT, 0.5 µM (A) and at least an order of magnitude higher than the influx K_t for PCFT-WT, 50 µM (5 µM for pemetrexed) (B). Transport at the high concentration reflects the influx V_max. Since a change in V_max should result in a comparable proportional change in influx, irrespective of the substrate concentration, a discrepancy in the percentage change between the low and high substrate concentrations reflects an additional change in the influx K_t. The data shown are percentages of influx activity in relation to PCFT-WT ± SEM from 3 independent experiments. *Comparing the differences between the percentage reduction in transport of MTX (P < .01), pemetrexed (not significant), and folic acid (P <. 01) at the low vs high concentrations. **Comparing the difference between 5-formylTHF and 5-methylTHF influx at the high concentration (P < .05). ***Comparing influx of MTX (P < .005) and folic acid (P < .005) with transport of pemetrexed at the low substrate concentration.

Figure 6.

Pemetrexed influx kinetics. Influx of 0.05 to 5.0 µM [³H]pemetrexed was assessed in cells transfected with either PCFT-WT or the N411K mutant. Data are best fit to the Michaelis-Menten equation V = V_max[S]/(K_t + [S]), where [S] is extracellular substrate concentration, V_max is the maximum transport rate, and K_t is the concentration at which influx is one half of maximum. Data shown are the means ± SEM from 3 independent experiments.

Figure 7.

Homology modeling of PCFT. Homology models of PCFT based on the reported structures of the bovine and rat fructose transporters, Glut5, in the inward open and outward open conformations, respectively.^, The 11th TMD with the Asn411 residue is highlighted. The predicted structure in the inward open configuration is indicated in blue. The predicted structure in the outward open conformation is indicated in green. (Left) A planar view of the protein. (Right) A view from the extracellular compartment into the aqueous translocation pathway. The Asn411 residue protrudes into the aqueous pathway and shifts further into the pathway when the protein is in the inward open conformation.